Thrombopoietin (TPO) is a platelet production regulation factor found in 1994 and is known to be composed of a glycoprotein having a molecular weight of 70-80 thousands produced mainly in liver. Thrombopoietin is a cytokine which in bone marrow promotes platelet precursor cells to survive, proliferate, differentiate and mature, namely promotes megakaryocytes to differentiate and proliferate. Thrombopoietin (TPO) receptor was identified earlier than TPO as c-Mpl, a receptor of a specific factor to regulate platelet production (M. Souyri et al., Cell 63: 1137 (1990)). It was reported that c-Mpl is distributed mainly in platelet precursor cells, megakayocytes and platelet cells and that the suppression of c-Mpl expression inhibits selectively megakaryocyte formation (M. Methia et al., Blood 82: 1395 (1993)). It was reported that the ligand to c-Mpl is TPO based on the results of proliferation assay of cells specific to c-Mpl ligand and purification of the ligand using c-Mpl (F. de Sauvage et al., Nature 369: 533 (1994); TD. Bartley et al., Cell 77: 1117 (1994)). At present Mpl is called TPO receptor. Therefore TPO and TPO receptor agonists have been expected to work as a therapeutic agent for thrombocytopenia, for example, as a medicine alleviating thrombocytopenia caused by bone marrow inhibition or bone marrow resection therapy for cancer patients.
On the other hand modified antibodies, especially antibodies with lowered molecular size, for example, single chain Fvs were developed to improve permeability into tissues and tumors by lowering molecular size and to produce by a recombinant method. Recently the dimers of single chain Fvs, especially bispecific-dimers have been used for crosslinking cells. Typical examples of such dimers are hetero-dimers of single chain Fvs recognizing antigens of cancer cells and antigens of host cells like NK cells and neutrophils (Kipriyanov et al., Int. J. Cancer, 77, 9763-9772, 1998). They were produced by construction technique of single chain Fv as modified antibodies, which are more effective in treating cancers by inducing intercellular crosslinking. It has been thought that the intercellular crosslinking is induced by antibodies and their fragments (e.g. Fab fragment), bispecific modified antibodies and even dimers of single chain Fvs, which are monospecific.
As antibodies capable of transducing a signal by crosslinking a cell surface molecule(s), there are known an antibody against EPO receptor involved in cell differentiation and proliferation (JP-A 2000-95800), an antibody against MuSK receptor (Xie et al., Nature Biotech. 15, 768-771, 1997) and others. There are also known an agonist antibody to TPO receptor, its fragments and single chain Fvs (WO99/17364). However there have been no reports on single chain Fv diners and modified antibodies such as single chain bivalent antibodies having agonist activity.
Noticing that single chain Fv monomers derived from monoclonal antibodies (antibody MABL-1 and antibody MABL-2 produced by the inventors) which induce apoptosis of LAP-containing cells do not induce apoptosis of cells and that dimers induce apoptosis, the inventors discovered that dimers crosslink (dimerize) LAP receptor on cell surface, thereby a signal is transduced into the cells and, as a result, apoptosis is induced. This suggests that monospecific single chain Fv dialers crosslink a cell surface molecule(s) (e.g. receptor) and transduce a signal like a ligand, thereby serving as an agonist.
Focusing on the intercellular crosslinking, it was discovered that the above-mentioned single chain Fv dimers do not cause hemagglutination while the above-mentioned monoclonal antibodies do. The same result was also observed with single chain bivalent antibodies (single chain polypeptides containing two H chain V regions and two L chain V regions). This suggests that monoclonal antibodies may form intercellular crosslinking while modified antibodies like single chain Fv dimers and single chain bivalent antibodies crosslink a cell surface molecule(s) but do not form intercellular crosslinking.
Based on those observations the inventors have newly discovered that modified antibodies such as single chain Fv dimers and single chain bivalent antibodies crosslink a cell surface molecule(s) or intracellular molecule(s) of the same cell, in addition to known intercellular crosslinking, and are suitable as a ligand to the molecule(s) (especially as a ligand which mimics the action of natural ligand).
Discovering further that an antibody molecule (whole IgG) can be modified into single chain Fv dimers, single chain bivalent antibodies and the like which crosslink a cell surface molecule(s), thereby reducing side effects caused by intercellular crosslinking and providing new medicines inducing only desired effect on the cell, the inventors completed the invention. The modified antibodies of the invention have remarkably high activity compared with whole antibodies (IgG) having the same V region as the modified antibodies. They have an improved permeability into tissues due to the lowered molecular size compared with antibody molecules and the lack of constant regions.